1. Field of the Invention
The present invention relates generally to the fields of neovascularization and cancer therapy. More particularly, it concerns the methods and compositions for treating cancer growth by first inhibiting angiogenesis and then employing radiotherapy.
2. Description of Related Art
Normal tissue homeostasis is achieved by an intricate balance between the rate of cell proliferation and cell death. Disruption of this balance either by increasing the rate of cell proliferation or decreasing the rate of cell death can result in the abnormal growth of cells and is thought to be a major event in the development of cancer. The effects of cancer are catastrophic, causing over half a million deaths per year in the United States alone. Conventional strategies for the treatment of cancer include chemotherapy, radiotherapy, surgery, biological therapy or combinations thereof; however further advances in these strategies are limited by lack of specificity and excessive toxicity to normal tissues. In addition, certain cancers are refractory to treatments such as chemotherapy, and some of these strategies such as surgery are not always viable alternatives.
Once the diagnosis of cancer is established, the most urgent question is whether the disease is localized, or has spread to lymph nodes and distant organs. The most fearsome aspect of cancer is metastasis, and this fear is well justified. In nearly 50% of patients, surgical excision of primary neoplasms is ineffective, because metastasis has occurred by the time the tumor is large enough for resection (Sugarbaker, 1977; Fidler and Balch, 1987). Metastases can be located in different organs and in different regions of the same organ, making complete eradication by surgery, radiation, drugs, or biotherapy difficult. Furthermore, the organ microenvironment significantly influences the response of tumor cells to therapy (Fidler, 1995), as well as the efficiency of anticancer drugs, which must be delivered to tumor foci in amounts sufficient to destroy cells without leading to undesirable side effects (Fidler and Poste, 1985). In addition, the treatment of metastatic cancer is greatly hindered due to the biological heterogeneity of cancer cells, and the rapid emergence of tumor cells that become resistant to most conventional anticancer agents (Fidler and Poste, 1985).
One of the processes involved in the growth of both primary and secondary (metastatic) tumors is neovascularization, or creation of new blood vessels which grow into the tumor. This neovascularization is termed angiogenesis (Folkman, 1986, 1989), which provides the growing tumor with a blood supply and essential nutrients. Although tumors of 1-2 mm in diameter can receive all nutrients by diffusion, further growth depends on the development of an adequate blood supply through angiogenesis. Inhibition of angiogenesis provides a novel and more general approach for treating both primary and secondary tumors by manipulation of the host microenvironment.
The induction of angiogenesis is mediated by several angiogenic molecules released by tumor cells, tumor associated endothelial cells and the normal cells surrounding the tumor endothelial cells. The prevascular stage of a tumor is associated with local benign tumors, whereas the vascular stage is associated with tumors capable of metastasizing. Moreover, studies using light microscopy and immunohistochemistry concluded that the number and density of microvessels in different human cancers directly correlate with their potential to invade and produce metastasis (Weidner et al., 1991, 1993). Not all angiogenic tumors produce metastasis, but the inhibition of angiogenesis prevents the growth of tumor endothelial cells at both the primary and secondary sites and thus can prevent the emergence of metastases.
Both controlled and uncontrolled angiogenesis are thought to proceed in a similar manner. Endothelial cells and pericytes, surrounded by a basement membrane, form capillary blood vessels. Angiogenesis begins with the erosion of the basement membrane by enzymes released by endothelial cells and leukocytes. The endothelial cells, which line the lumen of blood vessels, then protrude through the basement membrane. Angiogenic stimulants induce the endothelial cells to migrate through the eroded basement membrane. The migrating cells form a xe2x80x9csproutxe2x80x9d off the parent blood vessel, where the endothelial cells undergo mitosis and proliferate. The endothelial sprouts merge with each other to form capillary loops, creating the new blood vessel.
Persistent and unregulated angiogenesis is characteristic of tumor growth and it supports the pathological damage seen in these cancer. Thus, tumor growth is an angiogenesis-dependent process (Folkman, 1971). After an initial prevascular phase, every increase in tumor endothelial cell population is preceded by an increase in new capillaries converging on the tumor. Expansion of tumor volume beyond this phase requires the induction of new capillary blood vessels.
It has been demonstrated that in mice bearing Lewis lung carcinoma (3LL) subcutaneously (s.c.), the primary or local tumor releases an angiogenesis-inhibiting substance, named angiostatin (O""Reilly et al. 1994). Angiostatin is a 38-kDa fragment of plasminogen that selectively inhibits proliferation of endothelial cells. Angiostatin has been shown to suppresses vascularization and, hence, growth of metastases when used as an adjuvant to conventional therapy (U.S. Pat. No. 5,733,876, specifically incorporated herein by reference). Several studies have produced results consistent with this model. After systemic administration, purified angiostatin can produce apoptosis in metastases (Holmgren et al., 1995) and sustain dormancy of several human tumors implanted subcutaneously in nude mice (O""Reilly et al., 1996). However, although it is known that angiostatin can be generated in vitro from plasminogen by digestion with pancreatic elastase (O""Reilly, 1994), how it is generated in vivo in tumors remains unclear. Recently a second peptide, endostatin, was identified as a potential inhibitor of angiogenesis (O""Reilly et al., 1997). Endostatin, produced by hemangioendothelioma, is a 20 kDa C-terminal proteolytic fragment of collagen XVIII (Hohenester et al., 1998). Endostatin specifically inhibits endothelial proliferation and is postulated to inhibit angiogenesis and tumor growth.
Clearly, angiogenesis plays a major role in cancer development and maintenance. As stated earlier, conventional cancer therapeutic regimens are hampered by the ability of the cancer cell to adapt and become resistant to the therapeutic modality used to combat tumor growth. Although, it has been suggested that angiostatin may be useful in reducing the growth, size and otherwise mitigating the deleterious effect of a tumor, there is presently no objective evidence to suggest that angiostatin or endostatin could be used to weaken a tumor such that it would subsequently be amenable to radiotherapy.
Thus, the present invention provides a method of sensitizing a cell to ionizing radiation comprising the steps of first contacting the cell with an anti-angiogenic factor in an amount effective to sensitize the cell to ionizing radiation; and then exposing the cell to a dose of ionizing radiation effective to inhibit the growth of the cell.
In particularly preferred embodiments, the cell is an endothelial cell lining blood vessels that supply a tumor, defined hereafter as a tumor endothelial cell. In more defined embodiments, the tumor endothelial cell is located within an animal, and the contacting comprises in vivo delivery of the anti-angiogenic factor. In certain embodiments, the contacting is effected by direct injection of the tumor with the anti-angiogenic factor. In other embodiments, the contacting is effected by regional delivery of the anti-angiogenic factor. In still another embodiment, the contacting is effected by local delivery of the anti-angiogenic factor. In preferred embodiments, the anti-angiogenic factor may be delivered endoscopically, intratracheally, intralesionally, percutaneously, intravenously, subcutaneously or intratumorally.
In certain embodiments, the method may further comprise the step of tumor resection, prior to, after or during the contacting with the anti-angiogenic factor. In particularly defined embodiments, the anti-angiogenic factor is administered 2 days prior to the radiation. In other embodiments, the anti-angiogenic factor is administered 1 day prior to the radiation. In still another embodiment the anti-angiogenic factor is administered 12 hours prior to the radiation. In yet further embodiments, the anti-angiogenic factor is administered 6 hours prior to the radiation. In yet another embodiment, the anti-angiogenic factor is administered 3 hours prior to the radiation. In still another embodiment, the anti-angiogenic factor is administered 1 hour prior to the radiation. In yet another embodiment, the anti-angiogenic factor is administered 30 minutes prior to the radiation. In still another embodiment the anti-angiogenic factor is administered 15 minutes prior to the radiation. Another embodiment contemplates that the anti-angiogenic factor is administered immediately prior to the radiation. These are only exemplary embodiments, the time at which the anti-angiogenic factor is administered is only limited in the fact that it is administered prior to the administration of the radiation. Thus, the anti-angiogenic factor may be administered months, weeks, days, hours or minutes prior to the ionizing radiation. Further, the anti-angiogenic factor may be administered as a single dose, multiple doses over a measured period of time or as a continuous perfusion of the tumor. The time interval, time courses and doses of anti-angiogenic factor administration will depend on factors to be determined by the clinician. Such factors would take into account tumor size, other therapies being administered, condition of the patient and pharmacokinetic properties of the agent being administered.
The present invention provides methods of sensitizing cells to ionizing radiation by contacting the cells with an anti-angiogenic factor. In particular embodiments, the anti-angiogenic factor is angiostatin. In another embodiment, the anti-angiogenic factor is endostatin. In yet other embodiments, the cell is further sensitized to ionizing radiation by contacting the cell with the cytokine IL-12, in combination with an anti-angiogenic factor. In another embodiment, the cell is sensitized to ionizing radiation by contacting the cell with an antibody specific to VEGF, in combination with an anti-angiogenic factor.
In certain embodiments the tumor endothelial cell is a human tumor endothelial cell. In other more defined embodiments, the human tumor endothelial cell feeds a brain cancer cell. In other particularly preferred embodiments, the human tumor endothelial cell feeds a breast cancer cell. In certain embodiments, the tumor cell or tumor endothelial cell is resistant to anti-angiogenic factor therapy. In other embodiments, the tumor cell or tumor endothelial cell is resistant to radiotherapy.
In particular embodiments, the radiation employed may be ionizing radiation is X-radiation, xcex3-radiation, or xcex2-radiation.
In certain embodiments, contacting the cell with the anti-angiogenic factor comprises contacting the cell with an expression construct comprising an anti-angiogenic factor encoding gene operatively linked to a promoter, wherein the promoter directs the expression of the anti-angiogenic factor in the cell. In more defined aspects, the expression construct is selected from the group consisting of an adenovirus, an adeno-associated virus, a vaccinia virus and a herpes virus. In other defined aspects, the contacting comprises in vivo delivery of the expression construct.
In other aspects of the present invention the methods may further comprise the step of contacting the cell with a chemotherapeutic agent. In preferred embodiments, the chemotherapeutic agent is selected from the group consisting of adriamycin, 5-fluorouracil (5FU), etoposide (VP- 16), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP), doxorubicin, etoposide, verapamil, podophyllotoxin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin, daunorubicin, bleomycin, plicomycin, mitomycin, tamoxifen, taxol, transplatinum, vincristin, vinblastin and methotrexate.
Also contemplated by the present invention is a method for inhibiting growth of a cancer in a subject comprising the steps of first delivering to a cancer cell of the subject a therapeutically effective amount of a DNA molecule comprising a promoter operatively linked to an coding region that encodes an anti-angiogenic factor protein; and then exposing the cell to a dose of ionizing radiation; wherein the anti-angiogenic factor is delivered prior to the radiation and the expression of the anti-angiogenic factor sensitizes the cancer to the ionizing radiation and thereby inhibits the growth of the cancer.
In particularly defined embodiments, the promoter is a radiation responsive enhancer-promoter. It is contemplated that the delivering may be effected by regional delivery of the expression construct, local delivery of the expression construct, or direct injection of a tumor with the expression construct.
It is particularly contemplated that the contacting may comprise delivering the expression construct endoscopically, intratracheally, intralesionally, percutaneously, intravenously, subcutaneously or intratumorally.
In additional embodiments, the method may further comprise the step of tumor resection, prior to the contacting. Other embodiments contemplate the method as further comprising the step of tumor resection, after the contacting. In particularly defined embodiments, the cancer is selected from the group consisting of lung, breast, melanoma, colon, renal, testicular, ovarian, lung, prostate, hepatic, germ cancer, epithelial, prostate, head and neck, pancreatic cancer, glioblastoma, astrocytoma, oligodendroglioma, ependymomas, neurofibrosarcoma, meningia, liver, spleen, lymph node, small intestine, blood cells, colon, stomach, thyroid, endometrium, prostate, skin, esophagus, bone marrow and blood.
In other embodiments, the cancer is a solid tumor. In preferred embodiments, the tumor is a sarcoma. In other embodiments, the tumor is an epithelial tumor. In certain embodiments the cancer is a leukemia. In more defined embodiments, the leukemia is a promyelocytic leukemia.
In particular embodiments, the radiation responsive enhancer-promoter comprises at least one distal CArG domain of an Egr-1 promoter, a tumor necrosis factor xcex1 promoter or a c-jun promoter. In particular aspects, the CArG domain is from Egr-1 promoter. In other aspects the CArG domain is from tumor necrosis factor xcex1 promoter. In still other embodiments, the CArG domain is from c-jun promoter.
In specific embodiments, it is contemplated that the DNA molecule may further comprise a second coding region. The second coding region may encode a gene selected from the group consisting of a tumor suppressor, a cytokine, an enzyme, a receptor, or an inducer of apoptosis. Alternatively, the second coding region comprises an antisense construct. In preferred embodiments, the antisense construct is derived from an oncogene. More particularly, the oncogene may be selected from the group consisting ras, myc, neu, raf erb, src, fms, jun, trk, ret, gsp, hst, bcl and abl. In those embodiment in which the second coding region encodes a tumor suppressor, the tumor suppressor may selected from the group consisting of p53, p16, p21, MMAC1, p73, zac1, BRCAI and Rb. In those embodiment in which the second coding region encodes a tumor cytokine, the cytokine is selected from the group consisting of IL-2, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, TNF, GMCSF, xcex2-interferon and xcex3-interferon. In those embodiments in which the second coding region encodes an enzyme, the enzyme may be selected from the group consisting of cytosine deaminase, adenosine deaminase, xcex2-glucuronidase, hypoxanthine guanine phosphoribosyl transferase, galactose-1-phosphate uridyltransferase, glucocerbrosidase, glucose-6-phosphatase, thymidine kinase and lysosomal glucosidase. In preferred embodiment, the receptor may be selected from the group consisting of CFTR, EGFR, VEGFR, IL-2 receptor and the estrogen receptor. In other embodiment, the inducer of apoptosis is selected from the group consisting of Bax, Bak, Bcl-Xs, Bik, Bid, Bad, Harakiri, Ad E1B and an ICE-CED3 protease. In especially preferred embodiments, the subject is a human subject.
The present invention further provides a method of enhancing the effectiveness of ionizing radiotherapy comprising administering, to a tumor site in a mammal, an anti-angiogenic factor protein prior to radiation therapy; and ionizing radiation, wherein the combination of anti-angiogenic factor administration and radiation is more effective than ionizing radiation alone.
In preferred embodiments angiostatin is administered as a protein formulation. In particularly preferred embodiments the angiostatin is delivered as 25 mg angiostatin/kg body weight/day; 30 mg angiostatin/kg body weight/day; 35 mg angiostatin/kg body weight/day; 40 mg angiostatin/kg body weight/day; 50 mg angiostatin/kg body weight/day; 75 mg angiostatin/kg body weight/day; 100 mg angiostatin/kg body weight/day; 150 mg angiostatin/kg body weight/day; 200 mg angiostatin/kg body weight/day; 250 mg angiostatin/kg body weight/day; 300 mg angiostatin/kg body weight/day; 350 mg angiostatin/kg body weight/day; 400 mg angiostatin/kg body weight/day; 450 mg angiostatin/kg body weight/day; 500 mg angiostatin/kg body weight/day; 550 mg angiostatin/kg body weight/day; 600 mg angiostatin/kg body weight/day; 750 mg angiostatin/kg body weight/day; 800 mg angiostatin/kg body weight/day; 900 mg angiostatin/kg body weight/day or 1 g angiostatin/kg body weight/day. Of course, these are only exemplary concentrations and it is well within the skill of one in the art to modify these concentrations to arrive at a dose effective to sensitize cell to radiotherapy as described herein. In other preferred embodiments, the angiostatin is administered as part of a viral expression construct. In certain embodiments, the viral expression construct is in a composition comprising from about 108 to about 1010 virus particles.
In preferred embodiments endostatin is administered as a protein formulation. In particularly preferred embodiments the endostatin is delivered as 25 mg endostatin/kg body weight/day; 30 mg endostatin/kg body weight/day; 35 mg endostatin/kg body weight/day; 40 mg endostatin/kg body weight/day; 50 mg endostatin/kg body weight/day; 75 mg endostatin/kg body weight/day; 100 mg endostatin/kg body weight/day; 150 mg endostatin/kg body weight/day; 200 mg endostatin/kg body weight/day; 250 mg endostatin/kg body weight/day; 300 mg endostatin/kg body weight/day; 350 mg endostatin/kg body weight/day; 400 mg endostatin/kg body weight/day; 450 mg endostatin/kg body weight/day; 500 mg endostatin/kg body weight/day; 550 mg endostatin/kg body weight/day; 600 mg endostatin/kg body weight/day; 750 mg endostatin/kg body weight/day; 800 mg endostatin/kg body weight/day; 900 mg endostatin/kg body weight/day or 1 g endostatin/kg body weight/day. Of course, these are only exemplary concentrations and it is well within the skill of one in the art to modify these concentrations to arrive at a dose effective to sensitize cell to radiotherapy as described herein. In other preferred embodiments, the endostatin is administered as part of a viral expression construct. In certain embodiments, the viral expression construct is in a composition comprising from about 108 to about 1010 virus particles.
In still other preferred embodiments IL-12 is administered as a protein formulation. In particularly preferred embodiments the IL-12 is delivered as 0.1 pg IL-12/xcexcg protein/day; 0.5 pg IL-12/xcexcg protein/day; 1.0 pg IL-12/xcexcg protein/day; 5 pg IL-12/xcexcg protein/day; 10 pg IL-12/xcexcg protein/day; 15 pg IL-12/xcexcg protein/day; 20 pg IL-12/xcexcg protein/day; 25 pg IL-12/xcexcg protein/day; 30 pg IL-12/xcexcg protein/day; 35 pg IL-12/xcexcg protein/day; 40 pg IL-12/xcexcg protein/day; 45 pg IL-12/xcexcg protein/day; 50 IL-12/xcexcg protein/day; 55 pg IL-12/xcexcg protein/day; 60 pg IL-12/xcexcg protein/day; 65 pg IL-12/xcexcg protein/day; 75 pg IL-12/xcexcg protein/day; 80 pg IL-12/xcexcg protein/day; 85 pg IL-12/xcexcg protein/day; 90 pg IL-12/xcexcg protein/day; 95 pg IL-12/xcexcg protein/day; 100 pg IL-12/xcexcg protein/day; 200 pg IL-12/xcexcg protein/day; 300 pg IL-12/xcexcg protein/day; 400 pg IL-12/xcexcg protein/day; 500 pg IL-12/xcexcg protein/day; 600 pg IL-12/xcexcg protein/day; 700 pg IL-12/xcexcg protein/day; 800 pg IL-12/xcexcg protein/day; 900 IL-12/xcexcg protein/day; 1,000 pg IL-12/xcexcg protein/day; 2,000 pg L-12/xcexcg protein/day; 3,000 pg IL-12/xcexcg protein/day; 4,000 pg IL-12/xcexcg protein/day; 5,000 pg IL-12/xcexcg protein/day; 6,000 pg IL-12/xcexcg protein/day; 7,000 pg IL-12/xcexcg protein/day; 8,000 pg IL-12/xcexcg protein/day; 9,000 pg IL-12/xcexcg protein/day or 10,000 pg IL-12/xcexcg protein/day. Of course, these are only exemplary concentrations and it is well within the skill of one in the art to modify these concentrations to arrive at a dose effective to sensitize cell to radiotherapy as described herein. In other preferred embodiments, the IL-12 is administered as part of a viral expression construct. In certain embodiments, the viral expression construct is in a composition comprising from about 108 to about 1010 virus particles.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.